Uncovering accurate prognostic markers for high‐risk uveal melanoma through DNA methylation profiling

DEAR EDITOR Uveal melanoma (UM) is a rare, aggressive cancer with limited treatment options. Despite significant advancements in understanding its genetic background,1,2 the precise contribution of epigenomic alterations to the pathogenesis and progression of the disease remain elusive. In this study, we utilized a carefully curated set of UM samples to define the epigenomic and transcriptomic landscapes of high-risk tumours and identify novel, clinically relevant methylation markers and therapeutic targets. We stratified UM patients into risk groups based on UM-specific chromosomal rearrangements, particularly monosomy 3 (M3) and BAP1 mutations in tumour tissues (Figure 1A). Please refer to the Supporting information for a detailed description of patient clinical characteristics and methods (Additional file 1: Tables S1-S3). Specifically, we identified 25 low-risk and 33 high-risk patients, of which 21 (63.6%) carried the BAP1mutation (Figure 1B, C). We observed extensive gene expression reprogramming in high-risk UMs, resulting in 2262 differentially expressed genes (DEGs) including 60 epigenetic regulators, histone modifiers and chromatin remodelers. Furthermore, we identified 44 398 differentially methylated CpGs, with hypermethylation more frequent in TSS1500 and CpG shores (Supporting Information Additional file 1: Figures S1, S2; Additional file 2: Tables S1-4). Integrative analysis revealed 635 hypomethylated upregulated and 309 hypermethylated downregulated genes in highrisk tumours (Figure 2A,B). A significant proportion of methylation-regulated DEGs belong to specific functional groups, including epigenetic modifiers, transcription factors, tumour suppressor genes and oncogenes (Figure 2C), demonstrating the critical role of DNA methylation in controlling cell fate. The median β values of differentially methylated CpGs, were lower in the high-risk UMs, suggesting that epigenetic gene activation can be more common than repression (Figure 2D). BAP1 expression negatively correlated with cg01493712 DNA methylation


DEAR EDITOR
Uveal melanoma (UM) is a rare, aggressive cancer with limited treatment options. Despite significant advancements in understanding its genetic background, 1,2 the precise contribution of epigenomic alterations to the pathogenesis and progression of the disease remain elusive. In this study, we utilized a carefully curated set of UM samples to define the epigenomic and transcriptomic landscapes of high-risk tumours and identify novel, clinically relevant methylation markers and therapeutic targets.
We stratified UM patients into risk groups based on UM-specific chromosomal rearrangements, particularly monosomy 3 (M3) and BAP1 mutations in tumour tissues ( Figure 1A). Please refer to the Supporting information for a detailed description of patient clinical characteristics and methods (Additional file 1: Tables S1-S3). Specifically, we identified 25 low-risk and 33 high-risk patients, of which 21 (63.6%) carried the BAP1 mutation ( Figure 1B, C).
We observed extensive gene expression reprogramming in high-risk UMs, resulting in 2262 differentially expressed genes (DEGs) including 60 epigenetic regulators, histone modifiers and chromatin remodelers. Furthermore, we identified 44 398 differentially methylated CpGs, with hypermethylation more frequent in TSS1500 and CpG shores (Supporting Information Additional file 1: Figures S1, S2; Additional file 2: Tables S1-4). Integrative analysis revealed 635 hypomethylated upregulated and 309 hypermethylated downregulated genes in highrisk tumours (Figure 2A,B). A significant proportion of methylation-regulated DEGs belong to specific functional groups, including epigenetic modifiers, transcription factors, tumour suppressor genes and oncogenes ( Figure 2C), demonstrating the critical role of DNA methylation in controlling cell fate. The median β values of differentially methylated CpGs, were lower in the high-risk UMs, suggesting that epigenetic gene activation can be more common than repression ( Figure 2D). β value (r = −.496; p = .014), implying epigenetic control of BAP1 itself ( Figure 2E). Aberrant DNA methylation, distributed relatively uniformly across the entire genome, was associated with the dysregulation of key oncogenic pathways such as EGFR tyrosine kinase inhibitor resistance, focal adhesion, proteoglycans in cancer, PI3K-Akt signalling, or ECM-receptor interaction ( Figure 2F,G; Supporting Information Additional file 2: Table S5). These findings highlight the critical role of DNA methylation aberrancy in driving transcriptomic changes associated with poor prognosis.
Based on integrative analysis findings, we selected nine candidate genes, three upregulated and six downregu-  Figure S3) was significantly associated with UM overall survival (OS) data, available in The Cancer Genome Atlas dataset (Supporting Information Additional file 1: Figure S4). 3 The correlation between DNA methylation percentage measured by pyrosequencing in 58 UM tumours and β values were highly significant (Supporting Information Additional file 1: Table S4). In addition, individual DNA methylation values showed minimal overlap between high-and low-risk tissues (p < .001) ( Figure 3A), indicating the potential use of these markers for predicting risk groups with excellent diagnostic accuracy. AUC values ranged from .870 to .956 (p < .001) ( Figure 3B; Supporting Information Additional file 1:    respectively (p < .001), demonstrating the robustness and potential clinical utility of these epigenetic markers in UM risk stratification. Kaplan-Meier survival curves were generated with the log-rank test, and univariate Cox regression analysis was performed to confirm that DNA methylation of CALHM2 and MEGF10 genes and both methylation signatures were sufficient to stratify patients reasonably well as the standard risk groups based on chromosomal rearrangements and mutation profiling ( Figure 3C, Table 1).
The DNA methylation repatterning in UM was initially attributed to the loss of BAP1, a gene coding for a deubiquitinating hydrolase that exerts diverse functions such as cell cycle regulation, DNA damage repair, chromatin remodelling and gene expression control. 4 Although UM is considered poorly immunogenic due to its immuneprivileged site of origin, it has been proposed that BAP1 loss may promote the immunosuppressive tumour microenvironment (TME). 5 UM is a unique tumour type in which a high density of tumour-infiltrating lymphocytes and tumour-associated macrophages paradoxically correlates with a worse prognosis, highlighting the complex interaction between the TME and the immune response. Epigenetic regulations play a critical role in shaping these intricate relations. 6 Accordingly, six of the top nine methylation-regulated genes have been previously linked to immune functions. Specifically, EDNRB, IL12RB2, CALHM2 and RNF43, were identified among UM prognostic genes that interact with immune and stromal cells in the TME. 7 IL-12Rβ2, a subunit of the IL-12 receptor, generates high-affinity binding sites for IL-12, one of the most potent antitumor cytokines. 8 The prognostic significance of CALHM2 and RNF43 is further reinforced by their listing among the most important DEGs related to UM survival. 3 Additionally, AHNAK2, shown to promote UM cell proliferation and migration, 9 was found to correlate with infiltration of immune cell subpopulations such as CD8 + and CD4 + . 10 Our findings align with a recent report by Figueiredo et al., 5 which revealed that the downregulation of TLR1, a gene responsible for immune activation, is correlated with M3 status but not BAP1 expression, indicating epigenomic reprogramming independent of BAP1 mutations. These results highlight the importance of further exploring epigenetic regulation of the unique immune landscape of UM, which presents both challenges and opportunities for developing effective treatments for high-risk patients. Overall, our study provides compelling evidence for the substantial role of DNA methylation in UM progression by regulating the expression of genes involved in critical biological processes such as immune evasion, calcium homeostasis, adhesion and migration. Importantly, we demonstrate that the DNA methylation status of carefully selected CpG sites has the potential to serve as reliable prognostic biomarkers, underscoring the clinical relevance of DNA methylation analysis in UM. By leveraging the power of epigenetic profiling, we can gain a powerful tool for patient stratification, which can aid in personalized therapy and ultimately lead to improved outcomes.

A C K N O W L E D G E M E N T S
Foremost, we express our gratitude to all patients who consented to participate in this study. We would also like to thank Dr. Andrea Štanclova (Department of Molecular Biology and Genomics, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Bratislava, Slovakia) for her technical assistance and valuable contributions. Finally, we thank the Slovak Cancer Research Foundation for their enduring assistance and support.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare that they have no competing interests.